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Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacol 8:8

European Commission - Joint Research Centre, Institute for Environment and Sustainability, Rural, Water, and Ecosystem Resources Unit, Via E, Fermi 2749, 21027 Ispra (VA), Italy.
BMC Pharmacology 02/2008; 8:8. DOI: 10.1186/1471-2210-8-8
Source: PubMed

ABSTRACT A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue(R) using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene.
All four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints.
We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.

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    • "The interactions of anthropogenic chemical substances with the biota occur initially at the cellular level; hence, cellular responses not only are the first manifestations of toxicity but can also be used as appropriate tools for the early and sensitive detection of exposure to chemical substances (Fent, 2001). Cell viability analysis has been used in the field of ecotoxicology to evaluate the toxic effects caused by environmental pollutants (Bopp and Lettieri, 2008). Fish cell lines play an important role in toxicological research, serving as a model to study molecular mechanisms of toxicity and also as a test system for studying http://dx.doi.org/10.1016/j.tiv.2014.05.008 0887-2333/Ó 2014 Elsevier Ltd. "
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    • "The interactions of anthropogenic chemical substances with the biota occur initially at the cellular level; hence, cellular responses not only are the first manifestations of toxicity but can also be used as appropriate tools for the early and sensitive detection of exposure to chemical substances (Fent, 2001). Cell viability analysis has been used in the field of ecotoxicology to evaluate the toxic effects caused by environmental pollutants (Bopp and Lettieri, 2008). Fish cell lines play an important role in toxicological research, serving as a model to study molecular mechanisms of toxicity and also as a test system for studying http://dx.doi.org/10.1016/j.tiv.2014.05.008 0887-2333/Ó 2014 Elsevier Ltd. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transesterification has proved to be the best option for obtaining biodiesel and, depending on the type of alcohol used in the reaction, the type of biodiesel may be methyl ester or ethyl ester. Leaking biodiesel can reach water bodies, contaminating aquatic organisms, particularly fish. The objective of this study was to determine whether the soluble fraction of biodiesel (Bd), produced by both the ethylic (BdEt) and methylic (BdMt) routes, can cause cytotoxic, biochemical and genotoxic alterations in the hepatocyte cell line of Danio rerio (ZFL). The metabolic activity of the cell was quantified by the MTT reduction method, while genotoxic damage was analyzed by the comet assay with the addition of specific endonucleases. The production of reactive oxygen species (ROS) and antioxidant/biotransformation enzymes activity also were determined. The results indicate that both Bd increased ROS production, glutathione S-transferase activity and the occurrence of DNA damage. BdMt showed higher cytotoxicity than BdEt, and also caused oxidative damage to the DNA. In general, both Bd appear to be stressors for the cells, causing cytotoxic, biochemical and genetic alterations in ZFL cells, but the type and intensity of the changes found appear to be dependent on the biodiesel production route.
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